Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase is more commonly known by the abbreviation Rubisco. Rubisco is an enzyme participating in carbon fixation in the Calvin Cycle whereby atmospheric carbon dioxide is fixed and made available to biological systems in the form of energy-rich molecules.
In plants, algae, cyanobacteria and phototropic and chemoautotrophic proteobacteria, Rubisco comprises large subunit (LSU) chains and small subunit (SSU) chains. The substrate binding sites are located in the large chains. The large chains form dimers in which amino acids from each large chain contribute to the binding sites. A total of four large chain dimers and eight small chains assemble into a larger complex of about 540,000 Da.
Rubisco catalyzes the first step in photosynthetic CO2 assimilation (carbon reduction), as well as the competitive fixation of O2 which produces the waste product that is recycled in photorespiratory carbon oxidation. The importance of Rubisco is underlined by the fact that it is the major catalyst in the key chemical reaction by which inorganic carbon enters biological systems. Furthermore, Rubisco is a very abundant protein. Parry et al. (2003) suggest that Rubisco accounts for 30-50% of total soluble protein in chloroplasts.
However, the relative abundance of Rubisco may be attributable to the fact that it is a very slow acting enzyme, which only fixes a few CO2 molecules per second, in contrast to the thousands of chemical reactions per second characterizing many enzymes. The enzyme is inefficient as a catalyst for the carboxylation of RuBP and is subject to competitive inhibition by O2, to inactivation by loss of carbamylation, and to dead-end inhibition by RuBP binding prior to activation of the enzyme by carbamylation by CO2. This nonoptimal behavior makes Rubisco rate limiting for photosynthesis. Consequently, under most conditions and when light is not otherwise limiting photosynthesis, Rubisco is the primary rate-limiting enzyme of the Calvin Cycle.
As Rubisco is often rate limiting for photosynthesis in plants, improved forms of Rubisco would have considerable impact on increasing agricultural productivity. Several attempts have been made to increase the efficiency of Rubisco-mediated reactions. Previous approaches include introducing constructs which express Rubisco from one organism into another organism, increasing the level of expression of Rubisco subunits, expressing Rubisco small subunits from the chloroplast DNA, and altering Rubisco genes by mutagenesis so as to try to increase specificity for carbon dioxide (over oxygen) or otherwise increase the rate of carbon fixation.
Attempts have been made to introduce foreign Rubiscos, for example that from red algae such as Galdieria partita having a high CO2/O2 specificity, into flowering green plants. This would have been expected to improve the photosynthetic efficiency of crop plants, but these attempts failed due to problems with production, assembly and regulation of the foreign Rubisco in the host plant (Spreitzer and Salvucci, 2002; Parry et al., 2003). On the other hand, the large subunit of tobacco Rubisco has been successfully replaced with the homologous large subunit of the simpler purple photosynthetic bacterium Rhodospirillum rubrum which does not require a small subunit to fold and assemble into an active enzyme (Andrews and Whitney, 2003). While demonstrating that Rubisco replacement was achievable, the transgenic plant exhibited the very inferior specificity and catalytic properties of R. rubrum Rubisco.
Numerous attempts to define the roles of the active-site residues in specific steps of the reaction or to modify and improve the catalytic properties of Rubisco have been made using site-directed mutagenesis coupled with insights from X-ray crystallographic structures of Rubisco complexes from several species. However, these studies have failed to provide a detailed and self-consistent definition of the various roles of the residues of the active site over the complete reaction time course. These techniques have been uniformly unsuccessful in engineering a “better” Rubisco. While one mechanism by which Rubisco operates may be deduced by these studies, it may not be unique due to different possible interpretations of the incomplete experimental data, and, thus, it may not be the mechanism which exists in reality. For example, the mechanisms proposed in the Cleland consensus mechanism for Rubisco assume that a water molecule was displaced from the magnesium at the active site before formation of the reactive complex for carboxylation, and that consequently all subsequent steps in the reaction also proceed on the assumption that this displacement takes place. However, there is no experimental evidence that water is in fact displaced.
This contrasts with re-engineering programs for many other enzymes where single mutations, or in some cases multiple mutations, which were deduced straightforwardly from structural and mechanistic data obtained experimentally have proven successful in modifying substrate specificity or catalytic efficiency in predictable desired directions.
A major difficulty in implementing a rational re-engineering approach for Rubisco is that none of the reported experimental studies has provided direct evidence of the structure for all the intermediates involved, nor the precise roles of all the participating active-site residues, due to the aforementioned incompleteness of experimental data.
Experimental approaches are inherently unable to define precise roles for protons and water molecules involved in catalytic processes, as they are “invisible” to experimental probes. This difficulty is compounded by the complexity both of the Rubisco active site and by the fact that a sequence of reactions is involved. There is proposed to be a number of active-site “elements” comprising different combinations of active-site residues which take part in the different reactions steps, with the residue groups often being “reused”.
It appears that current Rubiscos represent only “partial evolutionary solutions” to optimizing the enzymic efficiency, i.e. that evolutionary processes have been unable to sample effectively the LSU sequence space, and that these current solutions represent far-from-optimum solutions. Thus, there is an opportunity to create more optimum solutions by a different route, or combination of routes, than biological evolution has been able to provide so far.
The creation or identification and introduction of more efficient forms of Rubiscos into photosynthetic organisms by transformation, selective breeding or other manipulations may allow more efficient growth of these organisms, including green plants and in particular flowering plants, as they would make more efficient use of water and nitrogen, and may grow more efficiently at higher temperatures. This in turn offers prospects for better yielding crops, the revegetation of degraded or drought-prone land, improved options for carbon sequestration and improvements in the production of biofuel or biomass energy and so on.
In summary, there is a need for generating proteins, such as a Rubisco, having improved functional properties wherein, for example, such proteins have improved efficiency and are adapted specifically for particular environmental conditions.